1. Volume 53, Issue 5, Pages 747-759 (March 2007)
Developmental Downregulation of Histone Posttranslational Modifications Regulates Visual Cortical Plasticity 
Elena Putignano, Giuseppina Lonetti, Laura Cancedda, Gianmichele Ratto, Mario Costa, Lamberto Maffei, Tommaso Pizzorusso 
Neuron 
Volume 53, Issue 5, Pages (March 2007)
DOI: /j.neuron
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1 Visual Stimulation Induces MSK Thr 581 Phosphorylation
(A) The number of pMSK-positive cells is increased by 15 min of visual stimulation. The activation is transient and returns to baseline at 40 min (dr, n = 7; 15 min, n = 11; 40 min, n = 4; one-way ANOVA, p = 0.003; post hoc Holm-Sidak: 15 min versus dr, p < 0.05; 15 min versus 40 min, p < 0.05; 40 min versus dr, p = 0.53). Hollow symbols represent data from single animals; filled symbols report average ± SEM. Data are normalized to the average of dr animals.
(B) Examples of pMSK staining in visual cortex of dr and 15 min mice. Cortical layers are indicated on the right. The inset shows the nuclear staining of pMSK as shown by a double staining for Neurotrace. Scale bar = 70 μm (10 μm for the inset).
(C) MSK phosphorylation induced by visual stimulation is blocked by inhibition of ERK. Average (±SEM) number of pMSK-positive cells (normalized to the dr value, dotted line) in visual cortex of mice visually stimulated for 15 min and treated with UO126. Significantly fewer positive cells are present in the treated cortex with respect to the contralateral untreated cortex (n = 6, paired t test, p = 0.01). The number of pMSK-positive cells in the cortex treated with the inhibitor vehicle is not different from that in the untreated contralateral cortex (n = 6, paired t test, p = 0.48). On the right, pMSK staining from a mouse visually stimulated for 15 min with one cortex infused with UO126. Scale bar = 60 μm.
(D) pMSK (red) and pERK (green) staining coexist in the same cells. Cells (n = 602) were classified as single-stained for pERK, single-stained for pMSK, or pMSK/pERK double-stained after inspection of z-stacks of confocal images. Scale bar = 14 μm.
Neuron  , DOI: ( /j.neuron )
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3. Figure 2
Visual Stimulation Induces Sustained CREB Ser 133 Phosphorylation
pCREB/CREB ratio (average ± SEM) was significantly increased by visual stimulation (one-way ANOVA, p < 0.05; post hoc Dunnett test, 40 min versus dr, p < 0.05; 15 min versus dr, p > 0.05; four samples for each experimental group).
Neuron  , DOI: ( /j.neuron )
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4. Figure 3 Visual Stimulation Induces H3 Ser 10 Phosphorylation
(A) Examples of pH3 staining in visual cortex of dr and visually stimulated mice. The inset shows the nuclear staining of pH3. Cortical layers are indicated on the left of the 40 min picture. Scale bar = 80 μm (10 μm for the inset).
(B) The number of pH3-positive cells is increased after 15 and 40 min of visual stimulation (dr n = 12, 15 min n = 10, 40 min n = 17; one-way ANOVA on ranks p < 0.001; post hoc Dunn's test: 15 min versus dr, p < 0.05; 40 min versus dr, p < 0.05; 40 min versus 15 min, p > 0.05). Hollow symbols represent data from single animals; filled symbols report average ± SEM. Data are normalized to the average of dr animals.
Neuron  , DOI: ( /j.neuron )
Copyright © 2007 Elsevier Inc. Terms and Conditions

5. Figure 4 Visually Induced H3 Phosphorylation Is ERK-Dependent
(A) H3 phosphorylation induced by visual stimulation is blocked by inhibition of ERK. The top graph shows the average (±SEM) number of pH3-positive cells in the visual cortex of mice visually stimulated for 15 and 40 min and treated with the MEK inhibitor UO126. Data are normalized to the dr value (dotted line). Gray bars represent data for the UO126-treated cortex (40 min, n = 7; 15 min, n = 5) and black bars represent values of the contralateral untreated cortex of the same animals (paired t test, p < 0.01 at both time points). The number of pH3-positive cells in the cortex treated with only the inhibitor vehicle (white bars in bottom graph; 40 min, n = 6; 15 min, n = 7) is not different from that of the untreated contralateral cortex of the same animals (black bars; paired t test, p = 0.89 at 40 min and p = 0.14 at 15 min). (Right) Examples of pH3 staining in the cortex infused with UO126 and in the contralateral untreated cortex from mice visually stimulated for 15 and 40 min. Scale bar = 70 μm.
(B) pH3 (red) and pERK (green) staining coexist in the same cells. Cells (n = 689) were classified as single-stained for pERK, single-stained for pH3, or pH3/pERK double-stained after inspection of z-stacks of confocal images. Scale bar = 15 μm.
Neuron  , DOI: ( /j.neuron )
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6. Figure 5 Visual Experience Induces ERK-Dependent H3 Acetylation
(A) Visual stimulation induces sustained H3 Lys 9 acetylation. The ratio between the intensity of the bands of AcH3 and total H3 was taken as an index of the amount of acetylated H3. TSA lane corresponds to NIH3T3 cells treated with TSA (positive control). The other lanes correspond to samples obtained from dr or from animals visually stimulated for 40 or 90 min. AcH3/H3 ratio was significantly increased by visual stimulation (one-way ANOVA on ranks p = 0.014; post hoc Dunn's test: 90 min versus dr, p < 0.05; 40 min min versus dr, p > 0.05; and 40 min versus 90 min, p > 0.05; dr, n = 6 samples; 40 min, n = 6 samples; 90 min, n = 7 samples; error bars = SEM).
(B) Histone Lys 9 acetylation induced by visual stimulation is blocked by the inhibitor of ERK activation SL237. Western blot of visual cortex samples of dr (n = 6) and visually stimulated mice treated with the ERK inhibitor (n = 5 samples) or vehicle (n = 5 samples). In control animals visual stimulation significantly increased the AcH3/H3 ratio with respect to dr, but this effect was completely prevented by SL237 (one-way ANOVA, p = 0.004; post hoc Holm-Sidak test: vehicle versus dr, p = 0.005; vehicle versus SL237, p = 0.002; dr versus SL237, p = 0.49; error bars = SEM).
Neuron  , DOI: ( /j.neuron )
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7. Figure 6
Comparison between Activation of ERK and CRE-Mediated Gene Expression in Juvenile and Adult Mice
For juvenile animals, we report data obtained in our laboratory and previously published in Cancedda et al. (2003). (A) Visual experience (15 min) caused ERK phosphorylation in the visual cortex of adult mice (dr, n = 4; adults, n = 7). Two-way ANOVA showed no effect of the age of the animals (p = 0.94) and a significant effect of visual experience (p < 0.001). Post hoc Hom-Sidak test (p < 0.05) showed that 15 min differed significantly from dr. Open symbols report single animal data; filled symbols report averages ± SEM. (B) Developmental downregulation of CRE-mediated gene expression induced by visual experience. In adult animals, 12 hr of visual experience caused only a modest increase of X-gal-positive cells compared with that in dr mice (t test, p < 0.05). The number of X-gal-positive cells in visually stimulated juvenile mice is much higher than in visually stimulated adult mice (t test, p < 0.01).
Neuron  , DOI: ( /j.neuron )
Copyright © 2007 Elsevier Inc. Terms and Conditions

8. Figure 7
Downregulation of Experience-Dependent Modifications of CREB and H3, but Not MSK, in Adult Mice
(A) Visual experience induces MSK phosphorylation in adult animals. Average number of pMSK-positive cells in visual cortex of adult (gray bars) mice in dr mice (n = 12) and in mice visually stimulated for 15 (n = 13) and 40 min (n = 11). Error bars = SEM. For comparison, the average of the pMSK-positive cells activated in juvenile mice (data of Figure 1) is reported. No difference is present between activation of pMSK in adult animals and in juvenile animals (Two-way ANOVA, effect of age p = 0.86; effect of duration of visual stimulation, p = 0.004; interaction time × age, p = 0.52; post hoc Holm-Sidak tests show that 15 min differs from both dr and 40 min mice, p < 0.03). (Right) Examples of pMSK staining in adult dr, dr + 15 min light, and dr + 40 min light. Scale bar = 70 μm.
(B) Reduced experience-dependent activation of H3 phosphorylation in adult animals. Average number (error bars = SEM) of pH3-positive cells in visual cortex of adult mice visually stimulated for 15 and 40 min or in dr (gray bars). One-way ANOVA shows a significant effect of visual stimulation (p = 0.029). Post hoc Holm-Sidak comparisons show that only the 15 min time point differs from dr (p = 0.02). For comparison, the average of the pH3-positive cells activated during the critical period (data of Figure 4) is reported. pH3 induction by visual experience is significantly different in adult and juvenile mice (two-way ANOVA: effect of age, p < 0.001; effect of duration of visual stimulation, p = 0.002; interaction time × age, p = 0.02; post hoc Holm-Sidak tests show that adults differ from juvenile mice both at 15 min and 40 min, p < 0.03). (Right) Examples of pH3 staining in adult dr, dr + 15 min light, and dr + 40 min light. Same calibration as in (A).
(C) Visual experience does not induce H3 acetylation in the adult visual cortex. The AcH3/H3 ratio (error bars = SEM) does not vary significantly after 40 or 90 min of visual experience with respect to dr (six samples for each group, one-way ANOVA, p = 0.41). The data for juvenile mice shown in Figure 6 are reported for comparison (white bars). (Right) Example of a western blot experiment on samples of adult visual cortex.
(D) Visual experience does not activate CREB phosphorylation in adult animals. pCREB/CREB ratio is not increased by visual stimulation at any time point (one-way ANOVA on ranks p = 0.375; nine samples for each group; error bars = SEM). For comparison, the time course of CREB phosphorylation observed in juvenile animals and shown in Figure 3 is reported. (Right) Example of a western blot experiment on samples of adult visual cortex.
Neuron  , DOI: ( /j.neuron )
Copyright © 2007 Elsevier Inc. Terms and Conditions

9. Figure 8
Inhibition of Histone Deacetylases Promotes Visual Cortical Plasticity in the Adult
(A) Example of a VEP response to stimulation of the contralateral eye.
(B) Example of localization of the receptive fields of the neurons generating the VEP response obtained in two tracks from the same nondeprived animal. In track 1, the response peaks with the stimulus positioned on the vertical meridian (i.e., 0° azimuth). In track 2, the response is maximal for stimuli placed more laterally in the visual field. Track 1 corresponded to a Contra/Ipsi ratio of 2.3; in track 2, Contra/Ipsi ratio was 3.6.
(C) TSA promoted ocular dominance plasticity in adult mice after 3 days of MD. Shaded area corresponds to the mean Contra/Ipsi ratio of the normal mice ± SEM (mean ratio = 2.19 ± 0.25, n = 8). Monocularly deprived mice receiving control treatment (Veh, n = 5) showed values similar to those of normal animals. In contrast, Contra/Ipsi ratio was significantly decreased in TSA-treated animals. (n = 6; one-way ANOVA, p = 0.007; post hoc Tukey test shows that TSA group is different from either the normal group [p = 0.012] or the VEH group [p = 0.016]. No difference is present between the normal group and the VEH group [p = 0.97]). For both groups recordings were performed from the cortex contralateral to the deprived eye. Hollow symbols report single animal data; filled symbols report the group average.
Neuron  , DOI: ( /j.neuron )
Copyright © 2007 Elsevier Inc. Terms and Conditions